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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
>chrX:134605508+134605607 100bp CTCATGACTATGCAGCGC CCCGCCTCATTCTTTCAAAC
Mut= 95 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
X-Linked
Sequence
Wt Sequence (deletion in lower case):
ATATGGCTCCTTTGAAAAACTTACTGATGTGAACTTGTAAAATAATAGTTTTCTTTGTTTCTCTTACTATATTATATACACTTTTTCATGACAATAGTTTTCTAATTTTTTTAAAGCCACATTGAATTTGCATTGAGCCAAGCTTGCCACCAAGAGTCCATCAGTCACCatgatgctgagcacagagggcagggaggggttcgtggtgaaggtcaggggcctaccctggtcctgctcagccgaggaagtgatgcgcttcttctctgattgcaaaatccaaaatggcacatcaggtgttcgtttcatctacaccagagaaggcagaccgagtggtgaagcatttgtcgaacttgaatctgaagatgaagtgaaattggctttaaagaaagacagagaaaccatgggacacagatatgttgaagtattcaaatccaacagtgttgaaatggattgggtgttaaagcatacaggtccaaatagtcctgatactgccaatgatggttttgtacgactcagaggactcccatttggctgtagcaaggaagaaattgttcagttcttttcagggttggaaattgtgccaaatgggatgacactgccagtggactttcaggggcggagcacaggggaagcttttgtgcagtttgcttcacaggagatagctgagaaggccttaaagaaacacaaggaaagaatagggcataggtacatcgaaatcttcaagagtagccgagctgaagtccgaacccactatgatccacctagaaagctcatgactatgcagcgcccgggtccttacgataggccaggggctggaagagggtataatagcattggcagaggagccgggtttgaaagaatgaggcggggtgcctatggtggagggtatggaggctatgatgactatggaggttataatgacgggtatggttttgggtctgatagatttggaagagacctaaactactgtttctcaggaatgtctgatcatagatatggagatggtgggtccagtttccagagcaccacggggcactgtgtacatatgagggggttaccatacagagccaccgagaatgatatttacaattttttctcacctcttaatcccatgagagtgcacattgaaataggacctgatggaagagttactggtgaggcagatgttgagtttgctactcatgaagacgctgtggcagctatggcaaaagataaagcaaatatgcaacacagatatgtggagctcttcttgaattctaccgcagggacaagtggaggagcttatgatcatagctatgtagagctctttttgaattctacagcaggggcaagtggtggtgcttacggtagccagatgatgggagggatgggtttatccaaccagtctagttatggaggtccggccagccagcagctgagtggtggctatggagggggttatggtggtcagagcagtatgagtggatatgaccaagttcttcaggaaaactcaagtgactatcagtcaaaccttgcttaggaagcaagggaccatccaataacatctacatgaataaaagctatctttataggaactggatagagtagggagggtgtctttatatccagtatgattggtaaATGGGAAGTACAACTGCTTCTGATCACTATTGGTCAGCTTCTTTCTTCTTTTTCCTTTTTTTTTTTTTAAAAAAAAGAACAAAAATTTAAGTTTTAACAGTTTCGCATTACAAGCTTGTGATTCATGCTTACTGTAAAGTGAAAGTTAAGATTGTTTTCAAACTTTAAGCTCAGCAATTTTGAACACTGAAAATATTCATCTAGGATGTAATAACAAGTTCAGTATTG
This mutation is a 1451 bp deletion beginning at Chromosome X position 134,604,908 bp and ending after 134,606,358 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 39357 | CTC ATG ACT ATG CAG CGC | Wild type Forward | A | |||
| 39358 | CCC GCC TCA TTC TTT CAA AC | Wild type Reverse | A | |||
| 39359 | TGA ATT TGC ATT GAG CCA AG | Mutant Forward | A | |||
| 39360 | GAA GAA AGA AGC TGA CCA ATA GTG A | Mutant Reverse | A | |||
| 39361 | Fluorophore-1 | AGA GGG TAT AAT AGC ATT GGC AGA | Quencher-1 | WT Probe | ||
| 39362 | Fluorophore-2 | ATC AGT CAC CAT GGG AAG TAC AA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 39357 | 0.40 uM |
| 39358 | 0.40 uM |
| 39359 | 0.40 uM |
| 39360 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.